Flavivirus ns1 subunit vaccine

ABSTRACT

The present invention relates to NS1 proteins or parts thereof of Flaviviruses, in particular of Dengue viruses useful for vaccination against said Flavivirus and against one or more other Flaviviruses. The invention further concerns the NS1 protein or parts thereof of one Dengue virus serotype, in particular serotype 2, useful for vaccination against Dengue viruses from all serotypes. The invention further concerns DNA comprising an expression cassette coding for a Flavivirus NS1 or parts thereof, vectors comprising said DNA and vaccines containing or expressing a Flavivirus NS1.

The present invention relates to NS1 proteins or parts thereof ofFlaviviruses, in particular of Dengue viruses useful for vaccinationagainst said Flavivirus and against one or more other Flaviviruses. Theinvention further concerns the NS1 protein or parts thereof of oneDengue virus serotype, in particular serotype 2, useful for vaccinationagainst Dengue viruses from all serotypes. The invention furtherconcerns DNA comprising an expression cassette coding for a FlavivirusNS1 or parts thereof, vectors comprising said DNA and vaccinescontaining or expressing a Flavivirus NS1.

BACKGROUND OF THE INVENTION

The etiological agent of the dengue fever is the Dengue virus, belongingto the Flavivirus genus of the family Flaviviridae (Burke and Monath,2001). A particularly important subgroup of Flaviviruses is the group ofso called mosquito-borne Flaviviruses, i.e. Flaviviruses that aretransmitted by mosquitos. This group comprises in addition to the abovementioned Denguevirus other important viruses such as the West nilevirus, the Japanese encephalitis virus and the Yellow fever virus(Fields Virology, ed. by Fields B. N., Lippincott-Raven Publishers,3^(rd) edition 1996, ISBN: 0-7817-0253-4, pages 931-1034). Typicaldiseases transmitted by these viruses are West nile fever and West nileencephalitis induced by the West nile virus, encephalitis induced by theJapanese encephalitis virus, Yellow fever induced by the Yellow fevervirus and Dengue fever, dengue hemorrhagic fever (DHF; see below) andDengue shock syndrome (DSS) induced by the Dengue virus.

Flaviviruses are enveloped, single-stranded, positive-sense RNA virusesformed by three structural proteins: the capsid protein (C) that forms anucleocapsid in association with the viral genome, which is surroundedby a lipid bilayer in which are anchored the M (membrane) and E(envelope) proteins. The genome is approximately 11 kb long and containsa single open reading frame encoding a polyprotein precursor of about3400 amino acid residues. Individual viral proteins are generated fromthis precursor by the action of cellular and viral proteases. The threestructural proteins (C, M and E) are derived from the N-terminal part ofthe polyprotein and are followed by seven non-structural proteins: NS1,NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Lindenbach and Rice, 2001).

Glycoprotein NS1, present in all Flaviviruses, appears to be essentialfor virus viability. Dengue virus NS1 is secreted from mammalianinfected cells in a soluble hexameric form (Flamand et al., 1999). Thisnoncovalently bound hexameric complex is formed by 3 dimeric subunitsand has a molecular mass of 310 kDa. Dimerization is a prerequisite forNS1 protein export to the plasma membrane, where it remains as theunique viral resident protein of the infected cell surface.

In mammalian cells, but not in insect cell lines that support dengueinfection, part of the transported NS1 is released into theextracellular milieu. Extracellular NS1 is secreted either as a solubleprotein, which exist in a higher hexameric oligomeric form, or inassociation with microparticles but not with virions. In addition, NS1has been found circulating in sera from dengue virus infected patients,suggesting that secretion of NS1 may be an important event in Flavivirusinfection in the human host. During the course of a Flavivirusinfection, the NS1 protein evokes a strong antibody response, whichhelps to clear the infecting virus from the host, presumably through acomplement-mediated pathway (Schlesinger, J. J. et al., 1987) andantibody-dependant cell cytotoxicity (ADCC) (Schlesinger, J. J. et al.,1993).

The Dengue virus, with its four serotypes Dengue virus serotype 1(Den-1) to Dengue virus serotype 4 (Den-4), is the most important memberof the Flavivirus genus with respect to infections of humans andproduces diseases that range from flu-like symptoms to severe or fatalillness, dengue haemorrhagic fever with shock syndrome. Dengue outbreakscontinue to be a major public health problem in densely populated areasof the tropical and subtropical regions, where mosquito vectors areabundant.

The concern over the spread of dengue infection and other diseasesinduced by mosquito-borne Flaviviruses in many parts of the world hasresulted in more efforts being made towards the development of denguevaccines, which could prevent both dengue fever (DF), and denguehemorrhagic fever (DHF) and in vaccines useful to protect the vaccinatedindividual against infections induced by some or all mosquito-borneFlaviviruses.

While most cases of DF are manifested after the first infection by anyof the four serotypes, a large percentage of DHF cases occur in subjectswho are infected for the second time by a serotype which is differentfrom the first infecting serotype of dengue virus. These observationsgive rise to the hypothesis that sequential infection of an individualwith antibody against one dengue serotype by a different virus serotypeat an appropriate interval may result in DHF in a certain number ofcases. Antibody-dependant enhancement (ADE) has been demonstrated invitro for dengue viruses, as well as other enveloped viruses, and isconsidered to be an important mechanism in the pathogenesis of DHF.

It has also been observed that DHF usually emerges in geographic areaswhere multiple (three or four) virus serotypes co-circulate. In regionswith endemic DHF such as Southeast Asian countries, the age-specificattack rate is higher in children, and the number of DHF cases decreasesin higher age groups. This roughly corresponds with the increasingseroprevalence to dengue, indicating that natural infection may evokeprotective immunity. This phenomenon is not unlike that observed withother viral infections such as hepatitis A virus. Anecdotal clinicalobservations have shown that patients may experience DHF twice(Nimmannitya et al., 1990) but this is rare, and it is difficult toidentify accurately the serotypes causing the second and subsequentinfections. So far, there has been no reports of a forth infection inthe same individual, despite the fact that all four dengue virusserotypes circulate in the same area. This suggests that, in nature,infection by two or three dengue virus serotypes in the same individualmay result in cross-reactive antibodies or even a cross-reactivecytotoxic lymphocyte response. This may modulate or protect againstinfection by the remaining dengue virus serotypes in nature.

At present there are no approved dengue vaccines. Today, prevention ofdengue virus infection is dependent upon control of the principalmosquito vector, Aedes aegypti. Insecticide resistance, lack oftechnical and financial support that would enable local healthdepartments to maintain effective mosquito control programs, andcontinuing geographic spread of both the vector mosquitoes and dengueviruses make it practically impossible to prevent dengue infections bycurrent mosquito control programs. Therefore, development of safe andeffective vaccines against all four serotypes of dengue virus has beendesignated by the WHO as a priority for the most cost-effective means toprevent dengue virus infection. The WHO has recommended that the idealvaccine against dengue and DHF should be the kind that can preventinfection caused by all serotypes so that sequential infection cannothappen.

To this end WO 98/13500 proposes to use a recombinant Modified VacciniaVirus Ankara (MVA) expressing antigens from all Dengue virus serotypesor to use four recombinant MVA wherein each of the recombinant MVAexpresses at least one antigen of one Dengue virus serotype. Bothstrategies provide very promising strategies to vaccinate against allDengue virus serotypes. However, it is desirable to provide a singlesubunit vaccine that upon administration results in an immune responseagainst more than one Flavivirus or against more than one serotype ofDengue virus, preferably against all Dengue virus serotypes. Moreover,WO 98/13500 discloses a recombinant MVA encoding Dengue virus NS1. WO98/13500 does not disclose that an antigen derived from one Dengue virusserotype elicits an immune response not only against the Dengue virusserotype from which the antigen is derived but also against antigensderived from other Dengue virus serotypes.

WO 99/15692 discloses a recombinant MVA containing and capable ofexpressing one or more DNA sequences encoding Dengue virus antigens notable to effect immune enhancement or antibody dependant enhancement. WO99/15692 does not disclose that an antigen derived from one Dengue virusserotype elicits an immune response not only against the Dengue virusserotype from which the antigen is derived but also against antigensderived from other Dengue virus serotypes.

OBJECT OF THE INVENTION

Thus, it is an object of the invention to provide a vaccine derived froma Flavivirus or a Flavivirus serotype that is stable, can easily beproduced and that leads to an immune response that protects thevaccinated individual not only against the Flavivirus or the Flavivirusserotype from which the vaccine is derived but also against otherFlaviviruses or Flavivirus serotypes. It is a particular object of thepresent invention to provide a vaccine derived from a mosquito-borneFlavivirus that protects the vaccinated individual not only against themosquito-borne Flavivirus or Flavivirus serotype from which the vaccineis derived but also against other mosquito-borne Flaviviruses orFlavivirus serotypes. It is a further object to provide a vaccine thatis derived from one Dengue virus serotype and that protects anindividual against an infection with at least two, preferably all Denguevirus serotypes.

DETAILED DESCRIPTION OF THE INVENTION

These objects have been solved using the NS1 protein or parts thereof ofa Flavivirus and DNA sequences comprising an expression cassette codingfor a Flavivirus NS1 protein or a part thereof, respectively. Inparticular, the object to provide a vaccine that is derived from onemosquito-borne Flavivirus and that protects an individual against aninfection with the mosquito-borne Flavivirus from which the vaccine isderived but also against an infection with at least one othermosquito-borne Flaviviruses has been solved by using the NS1 protein ora part thereof of a mosquito-borne Flavivirus, in particular the Denguevirus, preferably Dengue virus serotype 2 and corresponding DNAsequences, respectively. More specifically the object to provide avaccine that is derived from one Dengue virus serotype and that protectsan individual at least against an infection with at least two,preferably at least three, more preferably all Dengue virus serotypesand preferably also against the infection with other Flaviviruses, inparticular mosquito-borne Flaviviruses such as the Japanese encephalitisvirus, the Yellow fever virus and West Nile virus has been solved byusing the NS1 protein or a part thereof of a Dengue virus, in particularof Dengue virus serotype 2 and corresponding DNA sequences,respectively.

As it is shown in more detail in the experiment section the NS1 proteinderived from a Dengue virus of one serotype expressed de novo aftervaccination can evoke an antibody response that will cross react withNS1 proteins of Dengue virus serotype 1, 2, 3 and 4 plus NS1 from othermembers of the Flavivirus genus such as Japanese encephalitis virus,Yellow fever virus and West Nile virus. Thus, NS1 protein from oneDengue virus serotype origin is a universal DHF subunit vaccine forsimultaneous protection against at least two, more preferably three,even more preferably all four serotypes of dengue virus and furtheragainst one or more other viruses of the genus Flavivirus. Since in thissubunit vaccine strategy no E protein is involved, there should be norisk of Antibody Dependant Enhancement (ADE) upon subsequent exposure toany of the serotypes of dengue and therefore no vaccine related DHFshould be induced during natural outbreaks of dengue infection.

The NS1 protein may be expressed from a nucleic acid, preferably a DNAcomprising an expression cassette coding for at least a Flavivirus NS1protein or a part thereof. The term “at least” in this context is to beinterpreted in that the expression cassette may further encodeadditional proteins/peptides, either as separate proteins/peptides orfused to the NS1 protein or part thereof as defined in more detailbelow. In the context of the present invention the term “DNA” refers toany type of DNA, such a single stranded DNA, double stranded DNA, linearor circular DNA or DNA in the form of a plasmid or a viral genome. SinceFlaviviruses are RNA viruses the DNA coding for the Flavivirus NS1protein is a non-naturally occurring DNA, such as a cDNA or a syntheticDNA.

The term “expression cassette coding for a Flavivirus NS1 protein orpart thereof” is to be interpreted in that the coding sequence of aFlavivirus NS1 protein or a part thereof is preceded by elementscontrolling the transcription, in particular the initiation oftranscription. Examples for such transcriptional regulatory elements areprokaryotic promoters and eukaryotic promoter/enhancers. Preferredeukaryotic promoter/enhancers are the human Cytomegalovirus immediateearly promoter/enhancer and poxvirus promoters such as the 7.5 promoterand the poxvirus minimal promoter as disclosed in the example section.The sequence of the poxvirus minimal promoter is shown in FIG. 2 as wellas in SEQ:ID No. 9. The expression cassette may further contain elementscontrolling the termination of transcription such as prokaryotictermination elements or eukaryotic poly A signal sequences, ifnecessary.

The expression cassette may express only the NS1 protein or part thereofof a Flavivirus or may express the NS1 protein or part thereof togetherwith one or more further Flavivirus proteins/peptides, wherein the NS1protein or part thereof and the further proteins/peptides are producedas separate proteins/peptides or as fusion proteins/peptides. If notdefined otherwise in this description the term “peptide” in the contextof the present invention refers to a contiguous amino acid sequencestretch of at least 10 amino acids, more preferably of at least 20 aminoacids, most preferably of at least 25 amino acids.

The further Flavivirus protein is not the entire E-protein since thisprotein seems to be involved in the development of DHF. Thus, if thefurther Flavivirus peptide is derived from the E-protein it shouldcomprise less than 40 amino acids, preferably less than 35 amino acids.If amino acid sequence stretches derived from the E-protein areexpressed together with the NS1 protein or part thereof it should havebeen verified that this amino acid stretch does not comprise an epitopethat is involved in the generation of ADE and DHF.

If the expression cassette expresses in addition to the NS1 protein orpart thereof a further Flavivirus protein/peptide as separateproteins/peptides the expression cassette may comprise an Internalribosome entry site (IRES) between the sequence encoding the NS1 proteinor part thereof and the sequence encoding the further Flavivirusprotein. IRES elements are known to the person skilled in the art.Examples for IRES elements are the picornaviral IRES elements or the5″non-coding region of the hepatitis C virus.

Alternatively the nucleotide sequence encoding the NS1 protein or partthereof may be fused to a DNA sequence encoding further Flavivirusproteins/peptides in such a way that a fusion protein between the NS1protein or part thereof and the further Flavivirus protein/peptide isproduced. If the NS1 protein or part thereof and the further Flavivirusprotein/peptide are to be produced as fusion proteins/peptides therespective coding sequences are fused in frame.

In a preferred embodiment the DNA sequence encoding the NS1 protein orpart thereof is preceded by the sequence encoding the glycosylationsignal sequence of the E-protein. According to this embodiment a fusionprotein is produced that comprises the E-protein gylcosylation signalsequence fused to the NS1 protein or part thereof. As pointed out abovethe E-protein derived amino acid stretch should be as short as possibleand it should be excluded that this amino acid stretch contains anepitope involved in the generation of ADE and DHF. The gylcosylationsignal sequence of the E-protein fulfils these requirements.

In an alternative preferred embodiment the expression cassette usedaccording to the present invention contains as only Flavivirus sequencethe sequence encoding the NS1 protein or part thereof. Thus, in thispreferred embodiment the expression cassette according to the presentinvention does not express any other peptides/proteins from other partsof the Flavivirus genome, in particular not the NS2A or the E protein.

In a further alternative embodiment the DNA according to the presentinvention expresses the NS1 protein or part thereof as a fusion proteinwith proteins/peptides that are not derived from a flavivirus. Suchproteins/peptides comprise non-flaviviral signal sequences or sequencesthat are useful for the detection or purification of the expressedfusion protein, such as tags.

To understand the general structure of the Flavivirus sequence in theexpression cassette used according to a preferred embodiment of thepresent invention it is helpful to summarize briefly the genomestructure of Flaviviruses: During a natural Flavivirus infection thevirus produces a single polyprotein which is then cleaved first by hostcell proteases and then virus encoded proteases into the followingproteins: C, PrM and M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5(protein order along the polyprotein precursor). Therefore, a DNAsequence, in particular a cDNA sequence coding for the NS1 protein orpart thereof must require the addition of a “ATG” start codon. In apreferred embodiment the start ATG is then followed by a sequenceencoding a glycosylation signal so that the newly synthesized NS1protein becomes glycosylated in the endoplasmic reticulum. Such signalsequences are known to the person skilled in the art. Finally, theprotein-coding cassette needs a stop codon, which might be a TAG addedto the 3′ terminal end of the protein coding cDNA sequence. In theexample used in this invention the “ATG+signal sequence” element wasderived from the sequence encoding the hydrophobic C-terminal end of theE protein (the last 28 amino acids, which for the Dengue virus NewGuinea strain (“NGC strain”, GeneBank accession number AF038403) startswith the amino acid M (ATG). A typical expression cassette according tothe present invention is shown in FIG. 2 and as SEQ:ID No 9 and SEQ:IDNo. 10.

Thus, in summary this embodiment concerns the use of a DNA comprising anexpression cassette comprising the sequences coding for a Flavivirus NS1protein or part thereof, wherein the coding sequence is preceded by astart codon (“ATG”) and a sequence encoding a signal sequence forglycosylation, preferably derived from the E-protein as defined aboveand wherein the coding sequence is terminated by a stop codon oftranslation (FIGS. 1A, 1C and 2, SEQ:ID 5-10).

The DNA sequence that may be used according to the present inventionencodes a Flavivirus NS1 protein or part thereof. The term “Flavivirus”refers to any Flavivirus. More preferably the term “Flavivirus” refersto mosquito-borne Flaviviruses such as the West nile virus, the Japaneseencephalitis virus, the Yellow fever virus and the Dengue virus. The NS1protein or part thereof derived from one mosquito-borne virus encoded bya DNA according to the present invention should protect the vaccinatedindividual not only against an infection with the virus or virusserotype from which the vaccine is derived but also against theinfection with other mosquito-borne viruses or other serotypes of thevirus from which the vaccine is derived. The NS1 protein can preferablybe of any Dengue virus serotype. More preferably the NS1 protein codingsequence is derived from a Dengue virus serotype 2 such as the Denguevirus New Guinea strain (“NGC strain”, GeneBank accession numberAF038403). The terms “subtype” and “serotype” are used interchangeablythroughout this description.

The term “part thereof” in the context of the term “NS1 protein or partthereof” refers to an amino acid stretch of the NS1 protein, which issufficiently long to induce a specific immune response against the NS1protein from which the “part thereof” is derived. If the Flavivirus is aDengue virus the amino acid stretch should be an amino acid stretch thatprovokes an immune response in a vaccinated animal including a humanagainst the NS1 proteins of all Dengue virus serotypes. In the examplessection it is shown how the person skilled in the art can determinewhether an NS1 protein or part thereof induces an immune responsespecific for all Dengue virus serotypes. According to a preferredembodiment the Flavivirus DNA sequence encodes the entire NS1 protein.Thus, the term “NS1 protein or part thereof” relates to the entiresequence of naturally occurring NS1 proteins and shorter eptitopestretches that still elicit an immune response.

Moreover the term “NS1 protein” also relates to derivatives of naturallyoccurring NS1 proteins. Such a derivative may be a protein that has oneor more amino acid substitutions, deletions and/or insertion withrespect to the naturally occurring NS1 protein. By way of example such aderivative is a protein that has a homology in the amino acid sequenceof at least 50%, preferably of at least 75%, more preferably of at least90%. Consequently, the term “part thereof” also relates to parts of sucha NS1 protein derivative.

In summary one of the most preferred embodiments of the presentinvention is to use a DNA comprising an expression cassette coding for amosquito-borne Flavivirus NS1 or part thereof, wherein the Flavivirus ispreferably the Dengue virus, in particular Dengue virus serotype 2, andwherein the expression of the NS1 protein or part thereof is controlledby a transcriptional regulatory element. More preferably the DNAaccording to the present invention encodes the NS1 protein or partthereof as a fusion protein with a glycosylation signal sequence.

The invention further refers to vectors comprising a DNA as describedabove and to the use of said vectors to induce an immune responseaccording to the present invention. The term “vector” refers to anyvectors known to the person skilled in the art. A vector can be aplasmid vector such as pBR322 or a vector of the pUC series. Morepreferably the vector is a virus vector. In the context of the presentinvention the term “viral vector” or “virus vector” refers to aninfectious virus comprising a viral genome. In this case the DNA of thepresent invention is to be cloned into the viral genome of therespective viral vector. The recombinant viral genome is then packagedand the thus obtained recombinant vectors can be used for the infectionof cells and cell lines, in particular for the infection of livinganimals including humans. Typical virus vectors that may be usedaccording to the present invention are adenoviral vectors, retroviralvectors or vectors on the basis of the adeno associated virus 2 (AAV2).Most preferred are poxviral vectors. The poxvirus may be preferably acanarypox virus, a fowlpoxvirus or a vaccinia virus. More preferred ismodified vaccinia virus Ankara (MVA) (Sutter, G. et al. [1994], Vaccine12: 1032-40). A typical MVA strain is MVA 575 that has been deposited atthe European Collection of Animal Cell Cultures under the depositionnumber ECACC V00120707. Most preferred is MVA-BN or a derivative thereofwhich has been described in the PCT application WO 02/42480(PCT/EP01/13628). The content of this application is included in thepresent application by reference. MVA-BN has been deposited at theEuropean Collection of Animal Cell Cultures with the deposition numberECACC V00083008. By using MVA-BN or a derivative thereof the additionaltechnical problem has been solved to provide a particular safe virusvaccine against Flaviviruses since it has been shown that the MVA-BNvirus vector is an extremely attenuated virus. In particular, it hasbeen demonstrated that MVA-BN is more attenuated than the MVA strainsknown before in the prior art. MVA-BN is derived from Modified VacciniaAnkara virus and is characterized by the loss of its capability toreproductively replicate in human cell lines. MVA-BN is safer than anyother known vaccinia virus strains due to a lack of replication inhumans. In the preferred embodiment the invention concerns as a viralvector containing the DNA as defined above MVA-BN and derivatives ofMVA-BN. The features of MVA-BN, the description of biological assaysallowing to evaluate whether a MVA strain is MVA-BN or a derivativethereof and methods allowing to obtain MVA-BN or a derivative thereofare disclosed in WO 02/42480.

The term “derivatives” of the virus as deposited under ECACC V00083008,i.e. derivatives of MVA-BN, is used in the present application asdefined in WO 02/42480. In the following the features of a derivative ofMVA-BN are shortly summarized. For more detailed information regardingthe definition of a derivative of MVA-BN and in particular for detailedinformation regarding the biological assays used to determine whether aMVA virus is a derivative of MVA-BN reference is made to WO 02/42480.Thus, said term refers to vaccinia viruses showing at least one of thefollowing features of the deposited strain MVA-BN but showingdifferences in one or more parts of its genome. Preferably a derivativehas at least two, more preferably at least three, most preferably all ofthe following four features of MVA-BN:

-   -   capability of reproductive replication in chicken embryo        fibroblasts (CEF) and in the baby hamster kidney cell line BHK        (ECACC 85011433), but no capability of reproductive replication        in the human cell line HaCat (Boukamp et al. 1988, J Cell Biol.        106(3): 761-71),    -   failure to replicate in vivo,    -   induction of a higher immunogenicity compared to the known        strain MVA 575 (ECACC V00120707) in a lethal challenge model        and/or    -   induction of at least substantially the same level of immunity        in vaccinia virus prime/vaccinia virus boost regimes when        compared to DNA-prime/vaccinia virus boost regimes.

In particular a derivative of MVA-BN has essentially the samereplication characteristics than MVA-BN. Viruses having the same“replication characteristics” than the deposited virus are viruses thatreplicate with similar amplification ratios than the deposited strain inCEF cells and the cell lines BHK, HeLa, HaCat and 143B and that show asimilar replication in vivo as determined in the AGR129 transgenic mousemodel.

The term “not capable of reproductive replication” is used in thepresent application as defined in WO 02/42480. Thus, a virus that is“not capable of reproductive replication” is a virus that shows anamplification ratio of less than 1 in the human cell line HaCat (Boukampet al. 1988, J Cell Biol. 106(3): 761-71. Preferably, the amplificationrate of the virus used as a vector according to the invention is 0.8 orless in the human cell line HaCat. The “amplification ratio” of a virusis the ratio of virus produced from an infected cell (Output) to theamount originally used to infect the cells in the first place (Input)(“amplification ratio”). A ratio of “1” between Output and Input definesan amplification status wherein the amount of virus produced from theinfected cells is the same as the amount initially used to infect thecells.

In the context of the definition of MVA-BN and its derivatives the term“failure to replicate in vivo” is used in the present application asdefined in WO 02/42480. Thus, said term refers to viruses that do notreplicate in humans and in the mice model as explained in WO 02/42480.The mice used in WO 02/42480 are incapable of producing mature B- andT-cells (AGR 129 mice). In particular MVA-BN and its derivatives do notkill AGR129 mice within a time period of at least 45 days, morepreferably within at least 60 days, most preferably within 90 days afterthe infection of the mice with 10⁷ pfu virus administered intraperitonealy. Preferably, the viruses that show “failure to replicate invivo” are further characterized in that no virus can be recovered fromorgans or tissues of the AGR129 mice 45 days, preferably 60 days and zomost preferably 90 days after the infection of the mice with 10⁷ pfuvirus administered intra peritonealy.

MVA-BN and its derivatives are preferably characterized by a higherimmunogenicity compared to the known strain MVA 575 as determined in alethal challenge mouse model as explained in WO 02/42480. In such amodel unvaccinated mice die after the infection with replicationcompetent vaccinia strains such as the Western Reserve strain L929 TK+or IHD-J. The infection with replication competent vaccinia viruses isreferred to as “challenge” in the context of description of the lethalchallenge model. Four days after the challenge the mice are usuallykilled and the viral titer in the ovaries is determined by standardplaque assays using VERO cells. The viral titer is determined forunvaccinated mice and for mice vaccinated with MVA-BN and itsderivatives. More specifically MVA-BN and its derivatives arecharacterized in that in this test after the vaccination with 10²TCID₅₀/ml virus the ovary virus titers are reduced by at least 70%,preferably by at least 80%, more preferably by at least 90% compared tounvaccinated mice.

MVA-BN or its derivatives are preferably characterized by inducing atleast substantially the same level of immunity in vaccinia virusprime/vaccinia virus boost regimes when compared to DNA-prime/vacciniavirus boost regimes. A vaccinia virus is regarded as inducing at leastsubstantially the same level of immunity in vaccinia virusprime/vaccinia virus boost regimes when compared to DNA-prime/vacciniavirus boost regimes if the CTL response as measured in one of the “assay1” and “assay 2” as disclosed in WO 02/42480, preferably in both assays,is at least substantially the same in vaccinia virus prime/vacciniavirus boost regimes when compared to DNA-prime/vaccinia virus boostregimes. More preferably the CTL response after vaccinia virusprime/vaccinia virus boost administration is higher in at least one ofthe assays, when compared to DNA-prime/vaccinia virus boost regimes.Most preferably the CTL response is higher in both assays.

WO 02/42480 discloses how Vaccinia viruses are obtained having theproperties of MVA-BN and its derivatives as defined above.

Methods to insert the DNA as defined above into poxviral DNA and methodsto obtain recombinant poxviruses are known to the person skilled in theart. In a recombinant vaccinia virus the expression of the DNA accordingto the present invention is preferably, but not exclusively, under thetranscriptional control of a poxvirus promoter, more preferably of avaccinia virus promoter. The insertion of the DNA according to thepresent invention is preferably into a non-essential region of the virusgenome. In another preferred embodiment of the invention, theheterologous nucleic acid sequence is inserted at a naturally occurringdeletion site of the MVA genome (disclosed in PCT/EP96/02926).

In summary it is one of the most preferred embodiments of the presentinvention to provide a vector comprising the DNA as defined above,wherein the vector is MVA-BN or a derivative thereof and wherein the DNAcomprises an expression cassette coding for a Flavivirus NS1 protein orpart thereof, wherein the Flavivirus is preferably a Dengue virus, morepreferably Dengue virus serotype 2.

In a preferred embodiment the invention relates to the usefulness of theNS1 protein or part thereof encoded by a DNA according to the presentinvention or by a vector according to the present invention forvaccination against several flaviviruses or flavivirus serotypes. Forthe definition of the NS1 protein or part thereof according to thepresent invention reference is made to the above parts of thedescription where the DNA encoding NS1 has been defined by the productexpressed from said DNA. The following summary regarding the proteinaccording to the present invention is therefore not to be regarded as alimitation of the invention. In summary the NS1 protein can be anisolated NS1 protein or part thereof encoded by any Flavivirus. The NS1protein or part thereof is preferably derived from a Dengue virus, mostpreferably from Dengue virus serotype 2. The protein may only comprisethe amino acid sequence of a viral NS1 protein or part thereof. In apreferred embodiment the NS1 protein may contain additional amino acidsthat are required for an effective expression of the protein. Examplesfor such amino acids/amino acid sequences are shown above and includethe methionine at the N-terminus of the protein encoded by an added ATGcodon and an amino acid sequence derived from the C-terminal end of theE-protein acting as a signal sequence for glycosylation of the NS1protein or part thereof. Other signal sequences are also within thescope of the present invention. In an alternative embodiment the NS1amino acid sequence or a part thereof can be fused to otherproteins/peptides. Examples for fusion partners are sequences allowingthe identification of the protein such as tags or other flaviviralproteins or parts thereof.

In a preferred embodiment the present invention concerns the DNA, thevector or the NS1 protein or part thereof according to the presentinvention as a vaccine, in particular as a vaccine against severalflaviviruses or flavivirus serotypes. A “vaccine” is a compound, i.e. aDNA, a protein, a vector or a virus that induces a specific immuneresponse.

According to one alternative of this embodiment the “vaccine” accordingto the present invention is based on a Dengue virus NS1 protein or apart thereof which induces an immune response against the NS1 proteinsof all Dengue virus serotypes. In particular it has been shown that theNS1 protein of one Dengue virus serotype, in particular serotype 2,induces an immune response against the NS1 proteins of at least two,preferably at least three, most preferably all Dengue virus serotypesand preferably also against at least one other mosquito-borneFlavivirus.

As explained above the inventors of the present invention have foundthat the NS1 protein or part thereof according to the present inventionof one Flavivirus induces an immune response against the NS1 protein ofother Flaviviruses. As pointed out above the “Flavivirus” is preferablya mosquito-borne Flavivirus. In other words the inventors of the presentinvention have found that in an alternative embodiment the NS1 proteinor part thereof according to the present invention of one mosquito-borneFlavivirus induces an immune response against the NS1 protein of themosquito-borne Flavivirus from which the vaccine is derived and alsoagainst other mosquito-borne Flaviviruses. Thus, the vaccine derivedfrom a mosquito-borne Flavivirus is useful as vaccine against one ormore mosquito-borne flaviviruses. The term “vector derived from aFlavivirus” or similar terms in the context of the present descriptionmeans that a vector as defined above (e.g. a poxvirus vector or aplasmid) contains a DNA as defined above. Thus, this term refers to thevector insert and not the vector backbone. An example for a “vectorderived from a Flavivirus” is a poxvirus vector, such as MVA, comprisingan expression cassette comprising a poxvirus promoter, a sequenceencoding a Flavivirus NS1 protein or part thereof, wherein the sequencecoding for the Flavivirus NS1 protein or part thereof is preceded by anATG codon and a sequence encoding a glycosylation signal sequence andwherein the coding sequence is terminated by a stop codon oftranslation.

Thus the vaccination with the DNA, the vector or the NS1 protein or partthereof is useful as a single subunit vaccine against a broad range ofFlaviviruses or at least Flavivirus serotypes. The DNA or vectorencoding the NS1 protein or part thereof from one Flavivirus orFlavivirus serotype or the NS 1 protein or part thereof from saidFlavivirus or serotype can thus be used as a vaccine for vaccinationagainst other Flaviviruses and Flavivirus serotypes, respectively. Forexample a vaccine derived from a Dengue virus serotype 2 can be used asa vaccine against one, two or all of the serotypes 1, 3 and 4, as wellas vaccine against serotype 2. It may further be useful to protect anindividual against other Flaviviruses such as the West Nile Virus, theJapanese encephalitis virus and the Yellow fever virus.

In a preferred embodiment the DNA according to the present invention isused as a vaccine. It is known by the person skilled in the art that theadministration of naked DNA harboring a eukaryotic expression cassetteas in the present invention, in particular the intramuscular injectionof DNA leads to the expression of the protein encoded by the expressioncassette. The protein is exposed to the immune system and a specificimmune response is raised.

In an alternative embodiment the vaccination is made by administering avector according to the present invention, in particular a viral vector,more preferably a poxvirus vector, most preferably a vaccinia virusvector, e.g. a MVA vector.

For the preparation of vaccinia virus based vaccine, the virus accordingto the invention, in particular MVA-BN and its derivatives, is convertedinto a physiologically acceptable form. This can be done based on theexperience in the preparation of poxvirus vaccines used for vaccinationagainst smallpox (as described by Stickl, H. et al. [1974] Dtsch. med.Wschr. 99, 2386-2392). For example, the purified virus is stored at −80°C. with a titer of 5×10⁸ TCID₅₀/ml formulated in about 10 mM Tris, 140mM NaCl pH 7.4. For the preparation of vaccine shots, e.g., 10²-10⁹particles of the virus are lyophilized in 100 ml of phosphate-bufferedsaline (PBS) in the presence of 2% peptone and 1% human albumin in anampoule, preferably a glass ampoule.

It is particularly preferred that the vaccinia virus based vaccine, inparticular a MVA-BN based vaccine, used for vaccination is stored in afreeze-dried state. It is shown in the example section that the immunereaction as well as the percentage of cross reaction of the immuneresponse induced by the NS1 protein of one flavivirus to the NS1 proteinof different flaviviruses and flavivirus serotypes, respectively, isparticularly high if the virus used for vaccination was stored as freezedried virus. Thus, the vaccine shots preferably can be produced bystepwise freeze-drying of the virus in a formulation. This formulationcan contain additional additives such as mannitol, dextran, sugar,glycine, lactose or polyvinylpyrrolidone or other additives such asantioxidants or inert gas, stabilizers or recombinant proteins (e.g.human serum albumin) suitable for in vivo administration. An typicalvirus containing formulation suitable for freeze-drying comprises 10 mMTris-buffer, 140 mM NaCI, 18.9 g/l Dextran (MW 36000-40000), 45 g/lSucrose, 0.108 g/l L-glutamic acid mono potassium salt monohydrate pH7.4. After freeze-drying the glass ampoule is then sealed and can bestored between 4° C. and room temperature for several months. However,as long as no need exists the ampoule is stored preferably attemperatures below −20° C. For vaccination the lyophilisate can bedissolved in 0.1 to 0.5 ml of an aqueous solution, such a water,physiological saline or Tris buffer, and administered eithersystemically or locally, i.e. by parenterally, intramuscularly or anyother path of administration know to the skilled practitioner. The modeof administration, the dose and the number of administrations can beoptimized by those skilled in the art in a known manner. Most preferredfor poxvirus vectors is subcutaneous or intramuscular administration.Most preferably the vaccination is done by administration of two vaccineshots in an interval of e.g. 3 to 5 weeks.

If the vaccine is a MVA-BN vector or derivative thereof comprising a DNAaccording to the present invention a particular embodiment of thepresent invention concerns a kit for vaccination comprising a MVA-BNvirus vector according to the present invention for the firstvaccination (“priming”) in a first vial/container and for a secondvaccination (“boosting”) in a second vial/container.

If the vaccine is a MVA-BN vector or derivative thereof comprising a DNAas defined above a particular embodiment of the present inventionconcerns the administration of the vaccine in therapeutically effectiveamounts in a first inoculation (“priming inoculation”) and in a secondinoculation (“boosting inoculation”). The interval between the priminginoculation and the boosting inoculation is e.g. 2 to 12 weeks,preferably e.g. 3-6 weeks, more preferably e.g. about 3 weeks. The virusamount used for vaccination shout be at least 1×10² TCID₅₀, preferablye.g. 1×10⁷ TCID₅₀ to 1×10⁹ TCID₅₀. Moreover, a particular embodiment ofthe present invention concerns a kit for vaccination comprising a MVA-BNvirus vector as defined above for the first vaccination (“priming”) in afirst vial/container and for a second vaccination (“boosting”) in asecond vial/container.

Thus, the invention concerns in the vaccine embodiments a vaccinecomprising a DNA, a vector or a NS1 protein or part thereof as definedabove and the use of said DNA, vector or protein for the preparation ofa vaccine. According to a preferred embodiment the invention concernsthe use of said DNA, vector or protein for the preparation of a vaccinewherein the NS1 protein or part thereof, the NS1 protein or part thereofencoded by the DNA or the vector is from one Dengue virus serotype andwherein the DNA, the vector or the NS1 protein or part thereof is usedas a vaccine against two, three or all Dengue virus serotypes. Mostpreferably the Dengue virus serotype is serotype 2.

The invention further relates to a method for the treatment orprevention of a Flavivirus infection comprising inoculating an animal,including a human, in need thereof with a DNA as above, a vector asabove or a NS1 protein or part thereof as above. In particular theinvention relates to a method as above, wherein the NS1 protein or partthereof or the NS1 protein or part thereof encoded by the DNA or thevector is from one Dengue virus serotype and wherein the DNA, the vectoror the NS1 protein or part thereof is used as a vaccine against two,three or all Dengue virus serotypes

SUMMARY OF THE INVENTION

The invention relates in particular to the following, alone or incombination:

Use of

-   -   a nucleic acid comprising an expression cassette comprising a        transcriptional regulatory element and a sequence which codes at        least for the NS1 protein or a part thereof of a mosquito-born        flavivirus,    -   a vector comprising said nucleic acid and/or    -   a NS1 protein or part thereof of said flavivirus        for the preparation of a vaccine against the mosquito-borne        Flavivirus from which the nucleic acid or the NS1 protein or        part thereof is derived and against at least one other        mosquito-borne Flavivirus.        Use as above, wherein the mosquito-born Flavivirus from which        the nucleic acid or the NS1 protein or part thereof is derived        is a Dengue virus.

Use of a

-   -   nucleic acid comprising an expression cassette comprising a        transcriptional regulatory element and a sequence which codes at        least for the NS1 protein or a part thereof of a Dengue virus        serotype,    -   vector comprising said nucleic acid and/or    -   NS1 protein or part thereof of said Dengue virus serotype        for the preparation of a vaccine against all Dengue virus        serotypes and optionally against at least one other        mosquito-borne Flavivirus.        Use as above, wherein the Dengue virus from which the nucleic        acid or the NS1 protein or part thereof is derived is Dengue        virus serotype 2.        Use as above, wherein the sequence coding for the NS1 protein or        part thereof of the mosquito-borne flavivirus or of the Dengue        virus serotype is preceded by an ATG codon and a sequence        encoding a glycosylation signal sequence and wherein the coding        sequence is terminated by a stop codon of translation.        Use as above, wherein the other mosquito-borne Flavivirus is        selected from the West Nile virus, the Yellow fever virus and        the Japanese Enzephalitis virus.        Use as above, wherein the vector is a poxvirus vector.        Use as above, wherein the poxvirus vector is a Modified Vaccinia        Virus Ankara (MVA) strain, in particular MVA-BN deposited at the        European Collection of Cell Cultures under number V00083008 or a        derivative thereof.        Use as above, wherein the poxvirus vector is freeze-dried and is        reconstituted in a pharmaceutically acceptable diluent prior to        administration.        Use as above, wherein the transcriptional regulatory element is        a poxvirus promoter.        Use as above, wherein the vaccine is administered in        therapeutically effective amounts in a first inoculation        (“priming inoculation”) and in a second inoculation (“boosting        inoculation”)        Method for the treatment or prevention of a flavivirus        infections comprising inoculating an animal, including a human,        in need thereof with    -   a nucleic acid comprising an expression cassette comprising a        transcriptional regulatory element and a sequence which codes at        least for the NS1 protein or a part thereof of a mosquito-born        flavivirus,    -   a vector comprising said nucleic acid and/or    -   a NS1 protein or part of said flavivirus,        wherein the Flavivirus infection is an infection by the        mosquito-borne Flavivirus from which the nucleic acid, or the        NS1 protein or part thereof is derived and/or an infection by        another mosquito-borne Flavivirus.        Method as above, wherein the mosquito-borne Flavivirus from        which the nucleic acid or NS1 protein or part thereof is derived        is a Dengue virus.        Method for the treatment or prevention of a Flavivirus infection        comprising inoculating an animal, including a human, in need        thereof with    -   a nucleic acid comprising an expression cassette comprising a        transcriptional regulatory element and a sequence which codes at        least for the NS1 protein or a part thereof of a Dengue virus        serotype,    -   a vector comprising said nucleic acid and/or    -   a NS1 protein or part thereof of said Dengue virus serotype,        wherein the Flavivirus infection is an infection by the Dengue        virus serotype from which the nucleic acid, or the NS1 protein        or part thereof is derived and/or an infection by another Dengue        virus serotypes and/or an infection by other mosquito-borne        Flaviviruses.        Method as above, wherein the Dengue virus from which the DNA or        the protein or part thereof is derived is Dengue virus serotype        2.        Method as above, wherein the sequence coding for the NS1 protein        or part thereof of the mosquito-borne flavivirus or of the        Dengue virus serotype is preceded by an ATG codon and a sequence        encoding a glycosylation signal sequence and wherein the coding        sequence is terminated by a stop codon of translation.        Method as above, wherein the vector is a poxvirus vector.        Method as above, wherein the poxvirus vector is a Modified        Vaccinia Virus Ankara (MVA) strain        Method as above, wherein the MVA strain is MVA-BN deposited at        the European Collection of Cell Cultures under number V00083008        or a derivative thereof.        Method as above, wherein the poxvirus vector is freeze-dried and        is reconstituted in a pharmaceutically acceptable diluent prior        to administration.        Method as above, wherein the transcriptional regulatory element        is a poxvirus promoter.        Method as above, wherein the poxvirus vector or the        pharmaceutical composition is administered in therapeutically        effective amounts in a first inoculation (“priming inoculation”)        and in a second inoculation (“boosting inoculation”)        Poxvirus vector harboring a DNA comprising an expression        cassette comprising a transcriptional regulatory element and a        sequence which codes at least for a Flavivirus NS1 protein or a        part thereof, wherein the poxvirus is Modified Vaccinia Virus        Ankara (MVA) strain BN deposited at the European Collection of        Cell Cultures under number V00083008 or a derivative thereof.        Poxvirus vector as above, wherein the Flavivirus is a        mosquito-borne Flavivirus, in particular a Dengue virus.        Poxvirus vector as above, wherein the Dengue virus is Dengue        virus serotype 2.        Poxvirus vector as above, wherein the sequence coding for the        Flavivirus NS1 protein or part thereof is preceded by an ATG        codon and a sequence encoding a glycosylation signal sequence        and wherein the coding sequence is terminated by a stop codon of        translation.        Poxvirus vector as above, wherein the transcriptional regulatory        element is a poxvirus promoter.        Poxvirus vector as above, wherein the poxvirus vector is        freeze-dried.        Poxvirus vector as above as a vaccine.        Pharmaceutical composition comprising a poxvirus vector as above        and a pharmaceutically acceptable carrier, diluent and/or        additive.        Poxvirus vector as above or pharmaceutical composition as above        for the treatment and/or prevention of a flavivirus infection,        wherein the poxvirus vector or the pharmaceutical composition is        administered in therapeutically effective amounts in a first        inoculation (“priming inoculation”) and in a second inoculation        (“boosting inoculation”)        Method for the treatment or prevention of a flavivirus infection        comprising inoculating an animal, including a human, in need        thereof with a vector as above or with the pharmaceutical        composition as above.        Cell, preferably a human cell, comprising a poxvirus vector as        above.        Use of the poxvirus vector as above for the preparation of a        vaccine to treat or to prevent a Flavivirus infection.        Kit for prime/boost immunization comprising a poxvirus vector as        above or a pharmaceutical composition as above for a first        inoculation (“priming inoculation”) in a first vial/container        and for a second inoculation (“boosting inoculation”) in a        second vial/container.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1A: The dengue NGC strain “signal sequence+NS1” cDNA protein codingsequence of the construct used as an example in this invention. Thestart of the NS1 gene in the natural context is indicated by an arrow.Important features are the addition of an ATG start codon and a stopcodon (in this example “TAG”). Nucleotide sequence numbers refer toposition in the NGC strain genome (Genbank accession number AF038403).The nucleotide and amino acid sequence in FIG. 1A corresponds to SEQ:IDNo. 5. The amino acid is separately shown as SEQ:ID No. 6.

FIG. 1B: Diagram of plasmid pAF7NS1 containing the dengue NGC strain“signal sequence+NS1” protein coding sequence.

FIG. 1C: Nucleotide sequence of the NS1 cassette within plasmid pAF7showing the primer binding sites for PCR amplification of this cassettewith oBN345 and oBN338. The nucleotide and amino acid sequence in FIG.1C corresponds to SEQ:ID No. 7. The amino acid is separately shown asSEQ:ID No. 8.

FIG. 1D: top: Kyte-Doolittle hydrophicity plot of dengue NGC strain NS1amino acid sequence (amino acid 776 to 1127 of dengue NCG polyproteinGenbank Accession AF038403). Values above zero=hydrophobic. bottom:Kyte-Doolittle hydrophicity plot of dengue NGC strain NS1 amino acidsequence containing a signal sequence derived from the last 28 aminoacids of C-terminal of E protein (amino acid 748 to 775). The totalamino acid sequence represents amino acid 748 to 1127 of the dengue NCGpolyprotein (Genbank Accession AF038403) which for this strain startswith an “ATG” start codon but lacks a stop codon. Sig=Signal sequence.Values above zero=hydrophobic

FIG. 2: Nucleotide sequence of the “poxvirus promoter+signalsequence+NS1” expression cassette. The nucleotide and amino acidsequence in FIG. 2 corresponds to SEQ:ID No. 9. The amino acid isseparately shown as SEQ:ID No. 10. Briefly, the minimal poxvirusearly/late promoter element controls the expression of the NS1 proteinof Dengue virus serotype 2, wherein the N-terminus of the NS1 protein isfused to the 28 C-terminal aminoacids of the E-protein. The translationis terminated at an TAG stop codon that has been inserted into thenucleic acid sequence.

FIG. 3A: Cloning of NS1 expression cassette into the blunt ended Xho Isite (blunt end cloning) of pBNX07 to produce the clone pBN41.PPr=poxvirus promoter, D2F1=flank 1 of deletion site 2, NPT II=neomycinresistance gene, IRES=Internal Ribosome Binding Site, EGFP=EnhancedGreen Fluorescence Protein, NS1 (in pBN41)=signal sequence+NS1,D2F2=Flank 2 of deletion site 2, Sig=signal sequence. AmpR=Ampicillinresistance gene.

FIG. 3B: Hind III map of MVA (Genbank U94848) showing the location ofthe six deletion sites of MVA (-J-=junction of the deletion site). The“PPr+NPT II+IRES+EGFP+PPr+NS1” cassette was inserted into deletion 2site of MVA. PPr=poxvirus promoter, NPT II=neomycin resistance gene(protein coding sequence), IRES=Internal Ribosome Binding Site andNS1=signal sequence plus NS1 protein coding sequence of dengue 2 NGCstrain.

FIG. 4: Plot of the ELISA absorbance readings of the post-immunized seratitrations for all three rabbits.

FIG. 5: Elisa cross reactivity studies. The cross reactivity of a rabbitserum of day 38 (upper part) and day 66 (lower part) with lysates ofcells infected with DENV-1, DENV-3, DENV-4, JEV and WNV was tested in anELISA-assay.

EXAMPLES

The following examples will further illustrate the present invention. Itwill be well understood by a person skilled in the art that the examplesmay not be interpreted in a way that limits the applicability of thetechnology provided by the present invention to these examples.

Example 1 Construction of mBN07 1. Details of NS1 Antigen (FIG. 1)

The example refers to NS1 of serotype 2 derived from the New Guinea. Cstrain—NGC strain (example: Genbank sequence AF038403). Since the NS1protein of the Flaviviruses is produced as part of a polyproteinprecursor the NS1 gene in the corresponding DNA is not preceded by a“ATG” start codon.

Therefore, a cDNA sequence coding for the NS1 protein must require theaddition of a “ATG” start codon. This is then followed by the additionof a signal sequence so that the newly synthesized NS1 protein becomesglycosylated in the endoplasmic reticulum. Finally, the protein-codingcassette needs a stop codon and in this example TAG was added to the 3′terminal end of the protein coding cDNA sequence. In the example used inthis invention the “ATG+signal sequence” element was derived from thehydrophobic C-terminal end of the E protein (the last 28 amino acids,which for NGC strains starts with the amino acid M (ATG)).

FIG. 1A shows the exact signal sequence plus NS1 sequence used as theexample for this invention (see also SEQ:ID 5 and 6). The “signalsequence+NS1” nucleotide coding sequence was obtained by RT-PCRamplification from dengue NGC genomic RNA using the following primers:

D2NS 1-1 up: (SEQ: ID No. 4) 5′-ACAAGATCTGGAATGAATTCACGTAGCACCTCA-3′In italics: Bgl II restriction endonuclease recognition site.Underlined is the start codon.

D2NS1-2down: (SEQ: ID No. 3) 5′-AATAGATCTCTACTAGGCTGTGACCAAGGAGTT-3′In italics: Bgl II restriction endonuclease recognition site.Underlined is the stop codon.

The RT-PCR amplification was carried out using the Titan One Tube RT-PCRkit from Roche Molecular Biochemical (Catalog number 1-939-823)following the instructions recommended by the manufacturer. However,essentially any commercial or non-commercial RT-PCR kit can be usedinstead.

The RT-PCR product can then be cloned into the BamHI site of anymultiple cloning site present in many of the commercial bacterialcloning plasmids available but in this example it was cloned into pAF7to give rise to clone to pAF7D2NS1—see FIGS. 1B and 1C for sequencedetails for pAF7D2NS1. FIG. 1D shows the hydrophobicity plots of NS1amino acid sequence and NS1 containing the added signal sequence fromthe C-terminal amino acid coding sequence of E protein. The shortN-terminal hydrophobic domain is indicative of a signal sequence.

2. Details of NS1 Expression Cassette (FIG. 2)

To express this “signal sequence+NS1” from a poxvirus vector such ascanarypox, fowlpox, vaccinia or MVA, a poxvirus promoter needs to beadded to the 5′ end of this cDNA. Poly adenylation signal sequences arenot required as all poxvirus synthesized RNAs are polyadenylated by avirally encoded enzyme that requires no polyA addition signal sequencefor carrying out this function. Any poxvirus promoter can be used forthe expression of this cassette. FIG. 2 and SEQ:ID No. 9 and 10 show thenucleotide sequence of the “poxvirus promoter+signal sequence+NS1”cassette used as the example in this invention.

For the example used in this invention the “signal sequence+NS1” wasfurther PCR amplified from the NS1 plasmid clone using the primersoBN338 and oBN345. oBN345 primer contains a nucleotide sequence of apoxvirus minimal promoter element 5′ to the target sequence within thecloning plasmid. Plasmid target sequence for oBN345 primer binding wasapproximately 40 nucleotides upstream of the signal sequence startcodon. This was to ensure the RNA transcript contain a stretch ofnon-protein coding sequence before the signal sequence ATG start codon.

PCR primers with Ps promoter:

oBN338: (SEQ: ID No. 1) 5′-TTGTTAGCAGCCGGATCGTAGACTTAATTA (30 mer)oBN345: (SEQ: ID No. 2) 5′-CAAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAACACGATAATACCATGG-3′ (Underlined nucleotides present the poxvirusminimal promoter sequence.)

Annealing temperature for the PCR amplification reaction for the firstfive cycles was calculated from the nucleotide sequence that binds tothe homologous sequence in the cloning vector of oBN345.

3. Integration of NS1 Expression Cassette into MVA (FIG. 3)

The PCR amplification product was blunt end cloned into the cut andblunt ended Xho I site of plasmid pBNX07 (see FIG. 3 a) to form plasmidpBN41 (see FIG. 3 a). pBN41 is the vector for integrating the “poxpromoter+signal sequence+NS1” cassette into deletion site 2 of MVA byhomologous recombination.

The essential features of pBN41 (see FIG. 3 a) are as follows:

-   -   Plasmid backbone is pBluescript SK-plus from Stratagene (Genbank        VB0078)    -   D2F1: Deletion 2 flank 1 homologous recombination arm. This        represents the nucleotide sequence from 20117 to 20717 of the        MVA Genbank sequence U94848.    -   PPr: Poxvirus promoter.    -   NPT II: Neomycin phoshotransferase protein coding sequence        (protein coding sequence of Genbank V00618).    -   IRES: Internal Ribosome Entry Sequence from encephalomyocarditis        virus (Jang et al., 1989, Genbank M16802).    -   EGFP: Enhanced Green Fluorescence Protein coding sequence        (protein coding sequence—nucleotide 675 to nucleotide 1394 of        Genbank sequence U57609)    -   NS1: “signal sequence+NS1” protein coding sequence from dengue        NGC strain.    -   D2F2: Deletion 2 Flank 2 homologous recombination arm. This        represents the nucleotide sequence from 20719 to 21343 of the        MVA Genbank sequence U94848.    -   AmpR: Ampicillin resistance gene of pBluescript        3.1 Insertion of Dengue “Pox Promoter+Signal Sequence+NS1” into        Deletion Site of MVA by Homologous Recombination        3.1.1 Integration into MVA Genome by Homologous Recombination

The above integration vector pBN41 is used to integrate the dengue NS1expression cassette plus also the reporter cassette (Pox promoter+NPTII-IRES-EGFP) into the MVA genome by homologous recombination betweenflank 1 and flank 2 arms of pBN41 and the homologous target sequenceswithin the MVA genome. This is achieved by transfecting the linearizedintegration vector into chicken embryo fibroblast (CEF) cells previouslyinfected with MVA at low multiplicity of infection (MOI, for example,0.01 infectious units per cell). At 48 hours post infection or when theinfection had reached confluency a viral extract is prepared and storedat −20° C. ready for selection and clone purification of desiredrecombinant MVA (rMVA).

3.1.2 Selection of rMVA and Clone Purification

The elimination of non-recombinant MVA (empty vector virus) and theamplification of rMVA is achieved by infection of confluent chickenembryo fibroblast (CEF) cells at low MOI in the presence of G418 (amountof G418 has to be optimize to determine the highest dose that dose notkill the CEF cells). Any virus that does not contain and integrated NPTII gene will not replicate in the presence of G418 added to the cellmaintenance medium. G418 inhibits DNA replication but since the CEFcells will be in the stationary non-replicating state they will not beaffected by the action of G418. CEF cells infected with rMVAs can bevisualized under a fluorescence microscope due to the expression of theenhanced fluorescent green protein.

Viral extracts from the homologous recombination step must be seriallydiluted and used to infect fresh CEF cells in the presence of G418 andoverlaid with low-melting point agarose. After 2 days of infection, theagarose-infected plates are observed under a fluorescent microscope forsingle foci of green infected cells. These cells are marked and agaroseplugs containing the infected foci of cells are taken and placed into1.5 ml microcentrifugation tubes containing sterile cell maintenancemedium. Virus is released from the agarose plug by freeze-thawing thetube three times at −20° C.

The best clone or clones are further clone purified under agarose untilby PCR analysis there is no signs of empty vector contamination (3 to 30rounds of clone purification). These clones are then amplified forfurther stringent testing for correct insertion configuration, sequenceverification of promoter-foreign gene cassette and expression analysisby RT-PCR. After these analysis only one clone was further amplifiedunder G418 selection to prepare a master stock for furthercharacterization and immunogenicity studies.

The recombinant MVA with the inserted dengue NS1 expression cassettedescribed in this invention was named mBN07. FIG. 3 b shows theconfiguration of inserted foreign sequence in mBN07.

4. Expression of Authentic NS1 by MVA

The expression of the NS1 protein from the recombinant MVA, mBN07, wasverified by standard western blot analysis under non-denaturingconditions. More particularly, NS1 expression was analyzed afterpurified mBN07 was used to infect mammalian tissue culture cells, forexample BHK-21 cells, at an MOI of 1.0 infectious unit per cell. Crudeprotein extracts were prepared from these infected cells 24-30 hoursafter infection where portions of these extract were mixed with SDS-PAGEgel loading buffer containing 2-mercaptoethanol (2-ME) or not containing2-ME. These samples plus protein extract from cells (mosquito cell line)infected with dengue NGC strain as a positive control wereelectrophoretically separated in a SDS-PAGE gel and then blotted ontonitrocellulose membrane. The membrane was probed with an anti-dengue NS1monoclonal antibody.

It was shown that NS1 expressed by mBN07 is recognized by theanti-dengue NS1 monoclonal antibody and forms the correct dimeric formsimilar to NS1 from dengue infected cells (compare unboiled mBN07without 2-ME lane with unboiled DEN2 without 2-ME lane). Moreover it wasshown that the dimeric form resolves out to the monomeric forms underdenaturing conditions (see boiled mBN07 with 2-ME lane).

The NS1 expressed in cells infected with mBN07 was also recognized bypooled convalescent patients' sera and with monoclonal antibodies thatcross-react to NS1 of all four serotypes of dengue in western blotanalysis. This demonstrates that NS1 expressed by mBN07 is immunogenic.

Throughout the example section mBN07 (sometimes also termed BN07) iseither stored in a liquid state (optionally frozen) or in a freeze-driedstate. To obtain a freeze-dried virus a virus containing solution isprepared that comprises 10 mM Tris-buffer, 140 mM NaCl, 18.9 g/l Dextran(MW 36000-40000), 45 g/l sucrose, 0.108 g/l L-glutamic acid monopotassium salt monohydrate pH 7.4. Said formulation is thenfreeze-dried. For reconstitution water is added to the freeze-driedpreparation.

Example 2 Cross Immunogencity of NS1 Expressed by mBN07 to NS1 of DengueViruses Other than Serotype 2 and to the NS1 of Japanese EncephalitisVirus (JEV) and West Nile Virus (WNV)

1. NS1 Expressed from mBN07: Reactivity to Patient Convalescent Sera

Tested was the possibility that NS1 expressed by mBN07 is recognized byconvalescent patient sera from individuals who have evidence of previousdengue virus infection. Serum from 68 individuals who have antibodiesagainst dengue virus envelope protein (by immunoblotting againstauthentic antigens prepared from Dengue serotypes 1 to 4) were selectedfor testing against immunoblot strips prepared from mBN07 infected cellextracts and MVA-GFP infected cell extracts as control. Theantigen-containing cell lysates were treated with sample buffer without2 mercaptoethanol and were not heated. Of the 68 individual sera tested,62 (91.2%) reacted with BN07 NS1 expressed by mBN07 in immunoblots.These sera were further analyzed for reactivity to NS1 of all 4 denguevirus serotypes as well as Japanese encephalitis virus (JEV). Theresults are shown in Table 1. Fifty-four of the sera reacted with NS1 ofall dengue virus serotypes and Japanese Encephalitis Virus (JEV), and 53of these 54 sera (98.2%) also reacted with NS1 expressed from mBN07.Seven (7) sera were specific for NS1 of at least one dengue virusserotype and did not react with NS1 of JEV. All these 7 sera alsoreacted with NS1 expressed by mBN07. Another 7 sera reacted only withNS1 of JEV and not NS1 of any dengue virus serotype, yet 2 (28.6%) ofthese JEV-specific sera also reacted with NS1 expressed by mBN07.

TABLE 1 BN07 NS1 BN07 NS1 NEGATIVE POSITIVE AUTHENTIC DEN NS1 0% (0/7)100% (7/7) POSITIVE AUTHENTIC DEN & JEV 1.85% (1/54) 98.15% (53/54) NS1POSITIVE AUTHENTIC JEV NS1 71.43% (5/7) 28.57% (2/7) POSITIVE Comparisonof antiserum reactions against authentic NS1 and NS1 expressed by mBN07.In brackets: Number of samples tested positive/Total number of samplestested. DEN = dengue, JEV = Japanese encephalitis virus.

The same 68 sera were also analyzed by reactivity against premembraneproteins. In the experience of the inventors, antibodies againstpremembrane are far more specific than antibodies against NS1 or E. Thuspatients who have been infected with dengue will produce antibodieswhich recognize dengue virus premembrane and not JEV premembrane andvice versa. An analysis along these lines will provide a betterprediction of the history of infection of individuals. Table 2 showsthat sera from 22 patients reacted with authentic dengue premembraneprotein alone thus suggesting that these 22 patients have been exposedto dengue virus only and not to JEV. All these 22 sera reacted with NS1expressed by mBN07. Another 22 patients had evidence of previousinfection with both dengue and JEV, and again all 22 sera also reactedwith NS1 expressed by mBN07. In this series there were also 21 patientswho had evidence of previous infection with JEV only (even though thesesera had cross-reactive antibodies against dengue E). Interestingly, 17of the 21 (82%) JEV responders reacted with NS1 expressed by mBN07.There were only 3 sera in the whole set that did not react withpremembrane protein of either dengue or JEV and only 1 of these reactedwith NS1 expressed by mBN07. The most likely reason for this is that theantibody titer is too low to be detected by immunoblotting.

TABLE 2 BN07 NS1 BN07 NS1 NEGATIVE POSITIVE AUTHENTIC DEN prM 0% (0/22)100% (22/22) POSITIVE AUTHENTIC DEN & JEV prM 0% (0/22) 100% (22/22)POSITIVE AUTHENTIC JEV prM 19.0% (4/21) 81.0% (17/21) POSITIVE PrMNEGATIVE 66.7% (2/3) 33.3% (1/3) Comparison of antiserum reactionsagainst authentic premembrane and BN07 NS1. In brackets: Number ofsamples tested positive/Total number of samples tested. DEN = dengue,JEV = Japanese encephalitis virus.

The data in table 2 also clearly shows that of the 6 sera which did notreact with NS1 expressed by mBN07, 4 were from individuals who had beenpreviously infected with JEV and not dengue. The remaining 2 had nodetectable antibodies to premembrane protein of either dengue or JEV andwere likely to have been of a low titer.

2. mBN07 Vaccination of Rabbits and Testing of Post-Immunization SeraAgainst Dengue Virus and Japanese Encephalitis Virus Immunoblots andELISA Assays

Three specific pathogen-free rabbits were immunized by subcutaneousroute according to the vaccination schedule as shown below. Each rabbitwas vaccinated with one vial of freeze-dried vaccine (1×10e8 TCID50 BN07freeze-dried vaccine) reconstituted to 1 ml with sterile water on day 0and then again on day 28. Blood samples were taken prior to firstvaccination (prebleed) and again 10 days after second vaccination.

Day 0 = prebleed followed by 1^(st) vaccination Day 28 = 2^(nd)vaccination Day 38 = blood sampling Day 56 = 3^(rd) vaccination Day 66 =blood sampling Day 112 = 50 ml blood withdrawal from each rabbit

2.1 Testing of Prebleed and Post-Immunization Sera Against DengueSerotype 2 Immunoblots

Dengue 2 virus antigens and antigens of uninfected C6/36 cells wereseparated by SDS PAGE under non-denaturing conditions. For immunoblotassays serum of day 38 (diluted 1:200) was used.The results clearly demonstrated that upon vaccination with mBN07 allthree rabbit produced anti-NS1 antibodies of high titres that crossreact with authentic NS1 produced from a dengue serotype 2 infection oftissue culture mosquito cells. Serum taken before vaccination did notreact to any of the dengue protein on the immunoblots.

Post-immunized serum of day 38 was titrated at 1:1000, 1:2000, 1:4000,1:10⁻⁴, 1:10⁻⁵, 1:10⁻⁶ and tested on immunoblot strips of Dengue 2 virusantigens and control strips of uninfected C6/36 cells separated by SDSPAGE under non-denaturing conditions. Endpoint titers for the threerabbit sera of day 38 was calculated to be 1:10 000. The endpoint titersfor the sera of day 66 were calculated to be 1×10⁵ in both theimmunoblot assay an the ELISA, respectively (data not shown).

Both, pre- and post-immunized serum were titrated at 1:10⁻², 1:10⁻³,1:10⁴, 1:10⁻⁵, 1:10⁻⁶, 1:10⁻⁷ and tested in indirect IgG ELISA. Thewells were coated with dengue 2 and uninfected C6/36 lysates at 1:250dilution.

TABLE 3 1:10⁻² 1:10⁻³ 1:10⁻⁴ 1:10⁻⁵ 1:10⁻⁶ 1:10⁻⁷ Rabbit Pre −0.0120.006 0.007 0.003 −0.002 0.001 #1 Post 0.623 0.127 0.02 0.004 0.001−0.003 Rabbit Pre −0.012 0 −0.001 −0.003 0 −0.001 #2 Post 0.402 0.06−0.007 0.008 −0.003 0.002 Rabbit Pre −0.008 0.03 −0.002 0.005 −0.002−0.002 #3 Post 0.907 0.224 0.038 0.011 −0.001 −0.003 ELISA absorbancereading for pre- and post-immunized serum from each rabbit at differentdilutions (Pre = pre-immune sera, Post = post immunization sera).

The titration results for post-immunized sera of each rabbit wereplotted as shown FIG. 4. The estimated endpoint titers for each rabbitpost-immunized serum are 1:1000.

2.2 Testing of Prebleed and Post-Immunization Sera Against DengueSerotype 1, 3, and 4, Japanese Encephalitis Virus and West Nile VirusImmunoblots

Each of the rabbit serum of day 38 was tested at 1:1000 dilution onimmunoblot strips of Dengue 1, 2, 3, 4 and JE virus antigens pluscontrol strips of uninfected C6/36 cells separated by SDS PAGE undernon-denaturing conditions. It was shown that each rabbitpost-immunization sera reacts with NS1 from dengue serotypes 1, 3 and 4as well as with NS1 on Japanese encephalitis immunoblots.

Each of the rabbit serum of day 66 was tested at 1:1000 dilution onimmunoblot strips of Dengue 1, 3, 4, WNV and JE virus antigens pluscontrol strips of uninfected C6/36 cells separated by SDS PAGE undernon-denaturing conditions. It was shown that each rabbitpost-immunization sera reacts with NS1 from dengue serotypes 1, 3 and 4as well as with NS1 on Japanese encephalitis virus and West nile virusimmunoblots.

To confirm the immunoblot assays Elisa cross reactivity assays wereperformed. The wells of microtiterplates were coated with DENV-1,DENV-3, DENV-4, JEV, WNV and uninfected cell lysates at 1:250 dilutions.The sera were sera from day 38 (FIG. 5A) and day 66 (FIG. 5B).

From the immunoblot assays as well as from the ELISA experiments it canbe concluded that antibodies elicited by Dengue virus NS1 are crossreactive with all other Dengue serotypes, the JEV and the WNV.

2.3. Conclusions

-   -   Rabbits immunized with mBN07 vaccine elicited antibodies that        recognize authentic Dengue virus serotype 2 NS1.    -   Very high immune response was observed where end points were        1:10⁴ and 1:10⁻³ in both immunoblot assays and ELISA        respectively.    -   Antibodies elicited in the rabbits cross-reacted with all the        other dengue serotypes (1, 3 & 4).    -   The antibodies also cross-reacted with NS1 from a heterologous        virus such as JEV and WNV.

3. Immunogenicity Studies in Mice

Female out-bred mice were immunized by the intraperitoneal route withmBN07 expressing Dengue virus NS1 in different amounts and schedules asshown below. mBN08, a MVA corresponding to mBN07 but not expressing NS1and PBS served as control. The serum of the mice was used to checkwhether the antibodies generated in the mice were able to react onWestern blots with NS1 proteins from the different flavivirus serotypesand from the Japanese enzephalitis virus, respectively. The sera fromthe control mice were negative in all experiments.The following groups were analyzed:

Interval No. of before Group mice 1^(ST) dose Interval 2^(ND) dosebleeding 1 9 1 × 10⁷ TCID₅₀ 4 weeks 1 × 10⁷ TCID₅₀ 3 weeks BN07 each(0.1 ml BN07 each (0.1 ml of vaccine diluted of vaccine diluted 1:5 inPBS/mouse) 1:5 in PBS/mouse) 2 9 1 × 10⁷ TCID₅₀ 4 weeks 1 × 10⁷ TCID₅₀ 3weeks BN07 freeze-dried BN07 freeze-dried vaccine each vaccine each(reconstituted in (reconstituted in 1.2 ml water per mouse) 1.2 ml waterper mouse) 3 10 1 × 10⁷ TCID₅₀ 3 weeks 1 × 10⁷ TCID₅₀ 4 weeks BN07freeze-dried BN07 freeze-dried vaccine each vaccine each (reconstitutedin (reconstituted in 1.2 ml water per mouse) 1.2 ml water per mouse) 410 1 × 10⁷ TCID₅₀ 4 weeks 1 × 10⁷ TCID₅₀ 4 weeks BN07 each (0.1 ml BN07each (0.1 ml of vaccine diluted of vaccine diluted 1:5 in PBS/mouse) 1:5in PBS/mouse) 5 10 1 × 10⁷ TCID₅₀ 4 weeks 1 × 10⁷ TCID₅₀ 4 weeks BN07freeze-dried BN07 freeze-dried vaccine each vaccine each (reconstitutedin (reconstituted in 1.2 ml water per mouse) 1.2 ml water per mouse)In the immunoblot experiments the following results were obtained:

NO. OF DENV-1 DENV-2 DENV-3 DENV-4 JEV MICE POSITIVE POSITIVE POSITIVEPOSITIVE POSITIVE GROUP TESTED (% POS) (% POS) (% POS) (% POS) (% POS) 19 5 (55.5%) 9 (100%) 3 (33.3%) 7 (77.7%) 5 (55.5%) 2 9 8 (88.8%) 9(100%) 8 (88.8%) 9 (100%) 5 (55.5%) 3 10 9 (90%) 10 (100%) 9 (90%) 10(100%) 8 (80%) 4 10 8 (80%) 10 (100%) 9 (90%) 9 (90%) 4 (40%) 5 10 10(100%) 10 (100%) 9 (100%) 10 (100%0 8 (80%)Very similar experiments have been obtained with Balb/c mice: Mice werevaccinated with two shots of 1×10⁸ TCID₅₀ BN07 (freeze-dried andreconstituted with water) at days 0 and 21. The sera were obtained atday 42. 100% of the sera reacted with Dengue virus 2 NS1, 100% of thesera reacted with NS1 proteins from all four Dengue virus serotypes and75 of the sera reacted with the NS1 protein of JEV. The results obtainedwith 1×10⁸TCID₅₀ non-freeze dried BN07 were as follows: 100% of the serareacted with NS1 from all four Denguevirus serotypes. The serarecognized NS1 from JEV not as good as the sera obtained from micevaccinated with freeze-dried BN07.

CONCLUSION

-   -   100% of the mice immunized with BN07 had antibodies to DENV-2        NS1 with very strong response.    -   The immune response of mice immunized with 1×10e7 TCID50 BN07        was as strong as the immune response of mice immunized with        1×10e8 TCID50 BN07 (data not shown)    -   The best cross-reactivity percentage were observed at with mice        immunized at 4 weeks interval and 4 weeks before bleeding.    -   Mice immunized with the BN07 freeze-dried vaccine were observed        to have much stronger response to NS1 compared to those        immunized with the non-freeze dried vaccine.

REFERENCES

-   Nimmannitya S, Kalayanaroo S, Nisalak A, and Innes B. 1990. Second    attack of dengue hemorrhagic fever. Southeast Asian Journal of    Tropical Medicine and Public Health, 21:699-   Burke D S and Monath T P., 2001, Flaviviruses. In Fields Virology,    Fourth Edition, Edited by David M Knipe and Peter M Howley.    Published by Lippincott Williams and Wilkins, Philadelphia. Pages    1043-1125.-   Flamand M, Megret F, Mathieu M, LePault, Rey F A and Deubel    V., 1999. Dengue Virus Type 1 Nonstructural Glycoprotein NS1 is    Secreted from mammalian cells as a soluble hexamer in a    glycsylation-dependent fashion. J. Virol., 73:6104-6110.-   Jang S K, Davies M V, Kaufman R J and Wimmer E., 1989. Initiation of    protein synthesis by internal entry of ribosomes into the 5′    nontranslated region of encephalomycarditis virus RNA in vivo. J.    Virol., 63:1651-60.-   Lindenbach B D and Rice C M., 2001, Flaviviruses and their    replication. In Fields Virology, Fourth Edition, Edited by David M    Knipe and Peter M Howley. Published by Lippincott Williams and    Wilkins, Philadelphia. Pages 991-1041.-   Schlesinger J J, Brandriss M W and Walsh E E., 1987. Protection of    mice against dengue 2 virus encephalitis by immunization with the    dengue 2 virus non-structural protein NS1. J. Gen. Virol., 68:853-7-   Schesinger J J, Foltzer M and Chapman S., 1993. The Fc portion of    antibody to yellow fever virus NS1 is a determinant of protection    against yellow fever encephalitis in mice. Virology. 192: 132-41    The techniques and procedures described in this description are    familiar to a skilled practitioner(s) in the art of molecular    biology and virology especially relating to Flavivirus virology and    genetic manipulation of poxviruses. The techniques and procedures    described can be found in detail in the following literature    resources:-   Molecular Cloning, A laboratory Manual. Second Edition. By J.    Sambrook, E. F. Fritsch and T. Maniatis. Cold Spring Harbor    Laboratory Press. 1989.-   Virology Methods Manual. Edited by Brian W J Mahy and Hillar O    Kangro. Academic Press. 1996.-   Molecular Virology: A Practical Approach. Edited by A J Davison and    R M Elliott. The Practical Approach Series. IRL Press at Oxford    University Press. Oxford 1993. Chapter 9: Expression of genes by    vaccinia virus vectors.-   Current Protocols in Molecular Biology. Publisher: John Wiley and    Son Inc. 1998. Chapter 16, section IV: Expression of proteins in    mammalian cells using vaccinia viral vector.-   Antibodies, A Laboratory Manual. By Ed Harlow and David Lane. Cold    Spring Harbor Laboratory Press. 1988.

1-35. (canceled)
 36. A method of inducing an immune response in ananimal comprising administering an MVA virus vector comprising anexpression cassette to the animal; wherein the expression cassettecomprises a transcriptional regulatory element and a sequence thatencodes an entire Flavivirus NS1 protein of Dengue virus serotype 2, ora part thereof; and wherein the MVA virus vector induces an immuneresponse in the animal against one or more Flavivirus, selected fromDengue virus serotype 1, Dengue virus serotype 3, Dengue virus serotype4, Japanese Encephalitis virus, and West Nile virus.
 37. The method ofclaim 36, wherein the transcriptional regulatory element is a poxviruspromoter.
 38. The method of claim 36, wherein the expression cassettefurther comprises a nucleic acid encoding the hydrophobic C-terminal endof the E-protein of a Flavivirus.
 39. The method of claim 36, whereinthe expression cassette comprises: transcriptional regulatory element;an ATG initiation codon; a sequence encoding a glycosylation signalsequence; a sequence encoding an entire NS1 protein, or a part thereof,of Dengue Virus Serotype 2; and a translation termination codon.
 40. Themethod claim 36, wherein the expression cassette encodes a proteincomprising the amino acid sequence set forth in SEQ ID NO:10.
 41. Themethod of claim 36, wherein the MVA virus vector is characterized bybeing capable of reproductive replication in chicken embryo fibroblasts(CEF) and in the baby hamster kidney cell line BHK, but not capable ofreproductive replication in vitro in the human keratinocyte cell lineHaCaT.
 42. The method of claim 36, comprising administering the MVAvirus vector in a first inoculation (“priming inoculation”) and in asecond inoculation (“boosting inoculation”).
 43. The method of claim 36,wherein the animal is a human.
 44. The method of claim 37, wherein theanimal is a human.
 45. The method of claim 38, wherein the animal is ahuman.
 46. The method of claim 39, wherein the animal is a human. 47.The method of claim 40, wherein the animal is a human.
 48. The method ofclaim 41, wherein the animal is a human.
 49. The method of claim 42,wherein the animal is a human.